Food grade bacteria for the removal of toxic compounds

ABSTRACT

The present invention relates to food-grade bacteria and methods for removing toxic compounds, including lead, cadmium, mercury, arsenic and pesticides, from contaminated environments or substances.

This application is a divisional of U.S. patent application Ser. No.14/390,685 filed Oct. 3, 2014, which in turn is a national stageapplication under 35 U.S.C. 371 of International Application No.PCT/CA2013/000328, filed 5 Apr. 2013, which in turn claims the benefitunder 35 U.S.C. 119(e) of U.S. Provisional Ser. No. 61/620,796, filedApr. 5, 2012, the contents of each of which are hereby incorporated byreference into the present disclosure.

FIELD OF THE INVENTION

The present invention relates to food grade bacteria for improvingdetoxification. More particularly, the present invention relates to foodgrade bacteria, or extracts thereof, and to methods of using food gradebacteria or extracts thereof to reduce uptake of ingested toxiccompounds and to methods of sequestering toxic compounds from theenvironment to which the food-grade bacteria is exposed to.

BACKGROUND OF THE INVENTION

Humans and animals in general, are exposed to many toxic compounds thatcontaminate the environment, food chain, water supply and various itemsthat are part of everyday life. These range in number, type and exposurefrom ingredients in toothpaste and shampoos to drugs and pathogens inwell-water. Amongst Canadian First Nation and Inuit populations,environmental toxins are risk factors for other highly prevalentdiseases, especially type 2 diabetes [Sharp D. Environmental toxins, apotential risk factor for diabetes among Canadian Aboriginals. Int JCircumpolar Health. 2009; 68(4):316-26]. A large over-the-counterconsumer market has arisen under the guise of ‘detox’, but most of theproducts have no rationale or clinical evidence to support their use.The concept of detox has great appeal to consumers, both thehealth-conscious and others concerned with the growing number of storiesin the media about pollution and diseases related to toxic substances.Thus, there is substantial interest in this area, few effective productsand a growing need.

The replenishment or boosting of the beneficial organisms throughadministration of probiotics has become feasible in Canada relativelyrecently, and has led to much interest amongst consumer and healthcareprofessionals. Indeed, probiotics are one of the fastest growing foodsegments in North America. However, gaining insight into the mechanismsby which indigenous microbes and exogenous probiotics affect the subjecthas been limited.

Probiotic Lactobacilli and bifidobacteria have been shown to help manageseveral gut pathologies. For example, U.S. Pat. No. 6,641,808 disclosingthe use of Lactobacilli for the treatment of obesity; U.S. Pat. No.5,531,988, discloses a mixture of an immunoglobulin and a bacterium,such as Lactobacilli or bifidobacterium or mixtures thereof, that may beused to treat diarrhea, constipation, and gas/cramps; U.S. Pat. No.6,080,401 discloses a combination of probiotics having Lactobacillusacidophilus and Bifidobacterium bifidus and herbal preparations foraiding in weight loss, and so forth.

The ability of probiotic products to ameliorate toxins has been muchless studied, but nevertheless has some foundation. For example,Lactobacilli and/or bifidobacteria have been found to alter thesubject's intestinal metabolic signature [Ndagijimana, M. Laghi L,Vitali B, Placucci G, Brigidi P, Guerzoni M E. Effect of synbiotic foodconsumption on human gut metabolic profiles evaluated by 1H nuclearmagnetic resonance spectroscopy. Int J Food Microbiol. 2009; 134:147-153]; bind to aflatoxin (Lactobacillus strains) [Hernandez-MendozaA, Garcia H S, Steele J L. Screening of Lactobacillus casei strains fortheir ability to bind aflatoxin B1. Food Chem Toxicol. 2009;47(6):1064-8]; and detoxify or bind and negate other mycotoxins (B.animalis) [Fuchs S, Sontag G, Stidl R, Ehrlich V, Kundi M, Knasmüller S.Detoxication of patulin and ochratoxin A, two abundant mycotoxins, bylactic acid bacteria. Food Chem Toxicol. 2008; 46(4):1398-407].

In summary, the problem associated with toxic compounds is real, and ofgrowing concern to consumers.

Heavy Metals

Heavy metal toxicity is one of the largest health risks in the 21stcentury. Consumption of lead and cadmium through environmental exposureand diet has been directly responsible for poor health outcomesincluding: impaired neurological function and loss of IQ, osteoporosis,lung and kidney cancer.

Heavy metals such as lead and cadmium are present in the naturalenvironment, and therefore many bacteria over time have developedmechanisms of resistance to these metals which generally includeactively precipitating and sequestering the metals intra/extra cellularor the active efflux of metals out of the cell cytoplasm. Non-food gradebacteria have been investigated for their use in sequestration anddetoxification of heavy metals and have shown success (JS Singh et al.Genetically engineered bacteria: An emerging tool for environmentalremediation and future research perspectives. Gene. July 2011. 40(1-2):1-9); Rajkumar et al. Potential of siderophore-producing bacteriafor improving heavy metal phytoextraction. Trends Biotechnol. March2010. 28 (3):142-149).

Mercury

Mercury is one of the most toxic substances known to man and itsconsumption by a subject is linked to poor health outcomes includingaltered neurological development in children. Yet, North Americans andEuropeans are estimated to consume 6.7 μg daily of inorganic mercury andmethylmercury (World Health Organization, 1991).

Mercury is present in the natural environment, and as such, manybacteria have adopted mechanisms of resistance to it, which generallyreduce mercury levels in the surrounding environment. Many non-foodgrade bacteria have been investigated for their use in sequestration anddetoxification of mercury and mercury compounds in the environment,however the application of food grade bacteria has not been demonstratedto date.

Arsenic

Arsenic is a metalloid element which commonly comes in two oxidationstates: arsenate (As V) and arsenite (As III). Arsenic is founddistributed globally often in the earth's crust, it is highly soluble inwater and is found in high concentrations in ground water. Arsenictoxicity has been linked to a number of cases and is known to causeorgan failure, cancer and death. Main routes of exposure is throughingestion via diet, often arsenic contaminated waters are used forirrigation of farmland resulting in accumulation of the metal in plantsand food.

Pesticides

Pesticides such as malathion and parathion fall into the class oforganophosphate compounds and act as cholinesterase inhibitors.Malathion is one of the most widely used pesticides in the U.S., andparathion use has recently been limited and is not used in manydeveloped nations due to high toxicity. However, produce imports stillconsistently detect levels of parathion on produce and it is used insome rare instances in North America.

Major routes of public exposure is through consumption via diet.Agricultural workers and industrial workers are at increased risk ofexposure through work place by absorption or inhalation if safetyprotocols not properly followed.

In view of the problems associated to the exposure of any of the abovetoxic compounds, it would be advantageous to provide for food gradebacteria that can sequester toxic compounds, including heavy metals,mercury, arsenic, pesticides, such as malathion and parathion, or acombination thereof, from the gastrointestinal tract of a subject toreduce the amount of the toxic compound available to be absorbed by thesubject, while detoxifying the toxic compounds directly reduces thetoxicity of toxic compounds available to be absorbed by the subject.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide for food-gradebacteria or extracts thereof for the removal and/or neutralization oftoxic products from an environment or from a substance to which thefood-grade bacteria is exposed to, that solve the deficiencies inherentin traditional detoxification treatments. The present invention providesmethods and uses of food grade bacteria for removal and/orneutralization of toxic products found in the internal environment ofanimals, in the environment to which the animal is exposed or insubstance ingested or to be ingested by the animals that may avoidadverse side effects, is reasonable in cost, and may be beneficial inreducing the risk of diseases related to said toxic products. Further,the present invention is relatively easy to manufacture and deliver to asubject.

It is an object of the present invention to provide for food gradebacteria, or extracts thereof, to detoxify and/or sequester toxiccompounds, including heavy metals, mercury, arsenic and pesticides, withthe application of reducing a subject's toxic compounds exposure anduptake.

As such, in one embodiment, the present invention provides food-gradebacteria or extracts thereof for removing of toxic compounds from asubstance or environment to which the food-grade bacteria is exposed to.

In one embodiment, the present invention provides for a compositioncomprising a food-grade bacteria and a suitable carrier, whereby thecomposition comprises an effective dose of the food-grade bacteria toremove a toxic compound from a substance or environment to which thefood-grade bacteria is exposed to.

In one embodiment of the composition of the present invention, thetherapeutically effective dose is at least about 1×10⁹ of the food-gradebacteria per milliliter or less of the suitable carrier.

In another embodiment of the composition of the present invention, thesuitable carrier is a carbohydrate-containing medium.

In another embodiment of the composition of the present invention, thecarbohydrate-containing medium is a milk-based product.

In another embodiment of the composition of the present invention, thetoxic compound is selected from the group consisting of lead, cadmium,mercury, arsenic, malathion and parathion.

In another embodiment of the composition of the present invention, thefood-grade bacteria are provided dead or live.

In another embodiment of the composition of the present invention, thefood-grade bacteria are provided as an extract.

In another embodiment of the composition of the present invention, thecomposition comprises a combination of two or more different species offood-grade bacteria.

In another embodiment of the composition of the present invention, thecomposition comprises a combination of two or more strains ofLactobacillus rhamnosus, Lactobacillus casei, Lactobacillus crispatus,Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillusplantarum, Lactobacillus reuteri, and Lactobacillus amylovorus.

In another embodiment of the composition of the present invention, thefood-grade bacteria is selected from the group of food-grade bacterialisted in Table 1 shown bellow. It is mentioned that a bacteria strainof interest is the Lactobacillus rhamnosus strain deposited, accordingto the Budapest Treaty, at CNCM (Collection Nationale de Cultures deMicroorganismes, 25 rue du Docteur Roux, Paris) on Mar. 5, 2013, underthe accession number CNCM 1-4719. This strain is also referred to as “DN116-060” or R37.

In another embodiment of the composition of the present invention, theenvironment is an aqueous environment.

In another embodiment, the present invention is a composition, thecomposition including food-grade bacteria, a carrier and an animal'sfeed, wherein the food-grade bacteria is capable of removing a toxiccompound from a substance or environment to which the food-gradebacteria is exposed to and the food-grade bacteria comprises a bacterialisolate selected from the group consisting of the food-grade bacterialisted in Table 1 or any combination thereof.

In one embodiment, the present invention is a method for reducing asubject uptake of toxic compounds consumed by the subject, the methodincluding administering to the subject an effective dose of a food-gradebacteria capable of sequestering the toxic compound consumed by thesubject.

In another embodiment, a method for removing a toxic compound from asubstance or environment which is contaminated or suspected of beingcontaminated with the toxic compound is provided, the method includingcontacting the substance or environment with food-grade bacteria capableof removing the toxic compound from the substance or the environment.

In one embodiment, the present invention is a method of reducing thetoxic effects of a toxic compound in a subject, the method including:administering to the subject a therapeutically effective amount of afood-grade bacteria capable of removing the toxic compound from asubstance or environment.

In one embodiment of the previous methods of the present invention thetoxic compound is selected from the group consisting of lead, cadmium,mercury, arsenic, malathion and parathion.

In another embodiment of the previous methods of the present inventionthe food-grade bacteria are provided dead or live.

In another embodiment of the previous methods of the present inventionthe food-grade bacteria are provided as an extract.

In another embodiment of the previous methods of the present inventionthe food-grade bacteria comprise a combination of two or more differentspecies of food-grade bacteria.

In another embodiment of the previous methods of the present inventionthe composition comprises a combination of two or more strains ofLactobacillus rhamnosus, Lactobacillus casei, Lactobacillus crispatus,Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillusplantarum, Lactobacillus reuteri, and Lactobacillus amylovorus

In another embodiment of the previous methods of the present inventionthe food grade bacteria are selected from the group of food-gradebacteria listed in Table 1.

In one embodiment, the present invention is a method of obtaining astrain of Lactobacillus capable of removing a toxic compound from anenvironment, the method including a step of mutagenesis or genetictransformation of the Lactobacilus.

In one embodiment, the present invention is a method for obtaining acell fraction capable of removing a toxic compound from an environment,including the steps of: a) culturing a Lactobacillus strain, and b)recovering the cell fraction from the culture in step a).

In one embodiment of the last two methods the toxic compound is selectedfrom the group consisting of lead, cadmium, mercury, arsenic, malathionand parathion. In another embodiment Lactobacillus is provided dead orlive. In another embodiment the Lactobacillus is provided as an extract.In another embodiment the Lactobacillus includes a combination of two ormore different strains. In another embodiment, the Lactobacillus isselected from the group of Lactobacilli listed in Table 1.

In one embodiment, the present invention is a use of a food gradebacteria for the removal of a toxic compound from a substance or anenvironment.

In one embodiment of the use of the food grade bacteria, the toxiccompound is selected from the group consisting of lead, cadmium,mercury, arsenic, malathion and parathion.

In another embodiment of the use of the food grade bacteria, thefood-grade bacteria are provided dead or live.

In another embodiment of the use of the food grade bacteria, thefood-grade bacteria are provided as an extract.

In another embodiment of the use of the food grade bacteria, the foodgrade bacteria are provided as a combination of two or more differentspecies of food-grade bacteria.

In another embodiment of the use of the food grade bacteria, the foodgrade bacteria are provided as two or more strains of Lactobacillusrhamnosus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillusfermentum, Lactobacillus johnsonii, Lactobacillus plantarum,Lactobacillus reuteri, and Lactobacillus amylovorus.

In another embodiment of the use of the food grade bacteria, thefood-grade bacteria is selected from the group of food-grade bacterialisted in Table 1.

In another embodiment, the present invention provides for a method forremoving a toxic compound from a substance which is suspected of beingcontaminated with said toxic compound comprising contacting thesubstance with food-grade bacteria or extract thereof capable ofremoving the toxic compound from the substance.

In another embodiment, the present invention provides for a method ofreducing the toxic effects of a toxic compound in a subject, the methodcomprising: administering to the subject a therapeutically effectiveamount of food-grade bacteria of Table 1 or any combination thereof.

In one embodiment, the present invention provides for a method ofobtaining a strain of Lactobacillus capable of removing a toxic compoundfrom an environment, the method includes a step of mutagenesis orgenetic transformation of the Lactobacilus.

In another embodiment, the present invention is a method for obtaining acell fraction capable of removing a toxic compound from an environment.The method, in one embodiment, includes the steps of: a) culturing aLactobacillus strain, and b) recovering the cell fraction from theculture in step a).

In one embodiment of the methods of the present invention, thefood-grade bacteria comprise a combination of two or more differentspecies of food-grade bacteria.

In one embodiment of the present invention, the food grade bacteria is aLactobacillus.

In one aspect of the present invention the toxic compound includes aheavy metal.

In another aspect of the present invention, the toxic compound includesa heavy metal and the food-grade bacteria comprise dead bacteria.

In another aspect of the present invention, the toxic compound includesa heavy metal and the food-grade bacteria comprise live bacteria.

In one another of the present invention, the toxic compound includes aheavy metal and the food-grade bacteria comprise a mixture of deadbacteria and live bacteria.

In another aspect of the present invention the heavy metal is cadmium.

In another aspect of the present invention the heavy metal is lead.

In another aspect of the present invention the toxic compound includesmercury.

In another aspect of the invention the mercury is inorganic mercury.

In another aspect of the invention the mercury is organic mercury.

In one aspect of the present invention, the toxic compound includesmercury and the food-grade bacteria comprise dead bacteria.

In one aspect of the present invention, the toxic compound includesmercury and the food-grade bacteria comprise live bacteria.

In one aspect of the present invention, the toxic compound includesmercury and the food-grade bacteria comprise a mixture of dead bacteriaand live bacteria.

In another aspect of the present invention the toxic compound includesarsenic.

In one aspect of the present invention, the toxic compound includesarsenic and the food-grade bacteria comprise dead bacteria.

In one aspect of the present invention, the toxic compound includesarsenic and the food-grade bacteria comprise live bacteria.

In one aspect of the present invention, the toxic compound includesarsenic and the food-grade bacteria comprise a mixture of dead bacteriaand live bacteria.

In another aspect of the present invention the toxic compound includes apesticide.

In one aspect of the present invention, the toxic compound includes apesticide and the food-grade bacteria comprise dead bacteria.

In one aspect of the present invention, the toxic compound includes apesticide and the food-grade bacteria comprise live bacteria.

In one aspect of the present invention, the toxic compound includes apesticide and the food-grade bacteria comprise a mixture of deadbacteria and live bacteria.

In another aspect of the present invention the pesticide is selectedfrom malathion or parathion.

In another aspect of the present invention, the toxic compound includesendotoxins.

In another aspect of the present invention, the toxic compound includesheterocyclic aromatic amines.

In another aspect of the present invention, the toxic compound includesacrylamide.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will become more fully understood from thedetailed description given herein and from the accompanying drawings,which are given by way of illustration only and do not limit theintended scope of the invention.

FIG. 1A is a graph illustrating the ability of food grade Lactobacillito remove lead (Pb) from a solution (error bars±SEM).

FIG. 1B is a graph illustrating the ability of food grade Lactobacillito remove cadmium (Cd) from a solution (error bars±SEM).

FIG. 2A is a graph illustrating the ability of food grade Lactobacillito remove lead (Pb) from a solution compared to E. coli (errorbars±SEM).

FIG. 2B is a graph illustrating the ability of food grade Lactobacillito remove cadmium (Cd) compared to E. coli (error bars±SEM).

FIG. 3A is a graph illustrating the ability of live and dead food gradeLactobacilli to remove lead (Pb) from a solution (error bars±SEM).

FIG. 3B is a graph illustrating the ability of live and dead food gradeLactobacilli to remove cadmium (Cd) from a solution (error bars±SEM).

FIGS. 4A-4C are TEM microphotographs of Lactobacillus rhamnosus R37incubated with a control buffer without the addition of metals (FIG.4A), lead (FIG. 4B), and mercury (FIG. 4C).

FIGS. 5A-5C are scanning electron micrographs of Lactobacillus rhamnosusR37 incubated with a control buffer without the addition of metals (FIG.5A), lead (FIG. 5B), and mercury (FIG. 5C).

FIG. 6A is a scanning electron micrograph of Lactobacillus rhamnosus R37(top) and a corresponding energy-dispersive X-ray spectrum of a portionof a cell not containing visible deposits.

FIG. 6B is a scanning electron micrograph of Lactobacillus rhamnosus R37(top) and a corresponding energy-dispersive X-ray spectrum of a portionof a cell containing visible deposits.

FIGS. 7A-7C are scanning electron microphotographs of Lactobacillusrhamnosus GR-1 incubated with lead (FIG. 7A), cadmium (FIG. 7B), and acontrol without the addition of metals (FIG. 7C).

FIGS. 8A-8D are flow cytometry analysis of Caco-2 cell line comparingviability vs. mortality of untreated cells (FIG. 8A), cells exposed tocadmium (FIG. 8B), cells exposed to Lactobacillus plantarum 14917T (FIG.8C), and cells exposed to Lactobacillus plantarum 14917T and thenexposed to cadmium (FIG. 8D).

FIG. 9A is a graph illustrating the growth of a number of Lactobacillispecies in Man Rogosa Sharpe (MRS) media having lead.

FIG. 9B is a graph illustrating the growth of a number of Lactobacillispecies in MRS media having cadmium.

FIG. 10A is a graph illustrating the ability of a food grade bacteriumof the present invention to remove Hg²⁺ from a solution having a 1 partper million (ppm) Hg²⁺ inoculum (error bars±SEM; * signifies significant(p<0.05) difference by an unpaired T-test).

FIG. 10B is a graph illustrating the ability of a food grade bacteriumof the present invention to remove Hg²⁺ from a solution having a 15 partper billion (ppb) Hg²⁺ inoculum (error bars±SEM; * signifies significant(p<0.05) difference by an unpaired T-test).

FIG. 11 is a graph illustrating the ability of a food grade bacterium ofthe present invention to remove organic mercury from a solution (errorbars±SEM; * signifies significant (p<0.05) difference by an unpairedT-test).

FIG. 12 is a graph illustrating the ability of live and dead food gradebacterium of the present invention to remove inorganic mercury from asolution (error bars±SEM; * signifies significant (p<0.05) difference byan unpaired T-test).

FIGS. 13A-13B are graphs illustrating variability of mercury resistancewithin a group of food grade bacteria of the genus Lactobacillus. FIG.13A illustrates growth of different strains of Lactobacillus casei in agradient of Hg²⁺ and FIG. 13B illustrates growth of different strains ofLactobacillus rhamnosus in a gradient of Hg²⁺.

FIG. 14 is a graph illustrating twenty-four hour time course of mercuryremoval by Lactobacillus rhamnosus R37 and GR-1 in HEPES-NaOHsupplemented with 1 μg/mL HgCl₂ incubated at 37° C.

FIGS. 15A-15B are graphs illustrating removal of mercury from solutionby a selection of Lactobacillus rhamnosus strains of increasedresistance (R) and strains of increased sensitivity (S) to mercury atHgCl₂ concentrations of 0.5 ppm (FIG. 15A) and 1 ppb (FIG. 15B).

FIG. 16 is a graph illustrating the ability of food grade bacteria andE. coli species to remove As (III) and As (V) from solution at startinginoculums of 10 ppm.

FIG. 17 is a graph illustrating the ability of food grade bacteria toremove As (III) from solution at a starting inoculums of 1 ppm. (Errorbars±SEM).

FIG. 18 is a graph illustrating the ability of Lactobacilli to remove As(III) from solution at starting inoculums of 100 ppm.

FIGS. 19A-19B are graphs depicting the ability of probiotic bacteria toremove malathion (FIG. 19A) and parathion (FIG. 19B) from solution.Starting inoculums for malathion and parathion are 5 μg and 0.5 μgrespectively. (Error bars±SEM).

FIG. 20 is a graph illustrating the ability of a probiotic bacterium toremove both malathion and parathion from solution simultaneously.Malathion original concentration was 5 μg while parathion was 0.5 μg.(Error bars±SEM).

FIGS. 21A-21B are graphs depicting the ability of food grade bacteriaand E. coli to remove malathion (FIG. 21A) or parathion (FIG. 21B) fromsolution. Starting inoculums of pesticides for malathion and parathionwas 10 mg/L and 3 mg/L respectively. (Error bars±SEM).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Also, unless indicatedotherwise, except within the claims, the use of “or” includes “and” andvice versa. Non-limiting terms are not to be construed as limitingunless expressly stated or the context clearly indicates otherwise (forexample “including”, “having” and “comprising” typically indicate“including without limitation”). Singular forms including in the claimssuch as “a”, “an” and “the” include the plural reference unlessexpressly stated otherwise.

The expression “food grade bacteria” refers to any bacteria, alive ordead, that have no harmful effect on human health or that have a GRAS(generally recognized as safe) status. Such bacteria maybe selected fromthe group consisting of Lactobacilli and Bacilli. Non-limiting examplesof food-grade bacteria particularly suitable for the purpose of thepresent invention are listed in Table 1.

The term “probiotic” as used in this document refers to food-gradebacteria which perform beneficial functions to subject organisms whenthey are present and alive in viable form in the subject organisms.

“Food production animal” is used herein to describe any animal that isprepared and used for human consumption. A food production animal canbe, but not limited to, a ruminant animal such as beef and dairy cattle,pigs, lamb, chicken, turkey or any other fowl, or aquatic animalsincluding shrimp, lobster or fish used for human consumption.

As used herein, the term “removing a toxic compound from a substance orenvironment” refers to a removal of one or more toxic compounds that canbe tested as described in at least one of the examples below.

“Subject” or “subjects” are used herein to describe a member of theanimal kingdom, including food production animals and humans.

The present invention also encompasses mutant strains or geneticallytransformed strains derived from a parent strain. These mutant orgenetically transformed strains can be strains wherein one or moreendogenous gene(s) of the parent strain has (have) been mutated, forinstance to modify some of its metabolic properties (e.g., its abilityto ferment sugars, its resistance to acidity, its survival to transportin the gastrointestinal tract, its post-acidification properties or itsmetabolite production). They can also be strains resulting from thegenetic transformation of the parent strain by one or more gene(s) ofinterest, for instance in order to confer to said geneticallytransformed strains additional physiological features, or to allow it toexpress proteins of therapeutic or vaccinal interest that one wishes toadminister through said strains. These strains can be obtained from astrain by means of the conventional techniques for random orsite-directed mutagenesis and genetic transformation of Lactobacilli,such as those described by Gury et al. (2004) or by Perea Vélez et al.,2007, or by means of the technique known as “genome shuffling” (Patnaiket al., 2002 and Wang et al., 2007).

A subject of the present invention is also cell fractions which can beobtained from a Lactobacillus strain. They are in particular DNApreparations or bacterial wall preparations obtained from cultures ofsaid strain. They may also be culture supernatants or fractions of thesesupernatants. By way of example, cell-free supernatant (CFS) of oneLactobacillus strain can be obtained using the method for obtaining aCFS from another Lactobacillus strain.

A subject of the present invention is also a method for obtaining a cellfraction, comprising the steps of:

a) culturing a Lactobacillus strain, andb) obtaining and/or recovering the cell fraction from the culture instep a).

In compositions of the invention, said strain can be used in the form ofwhole bacteria which may be living or dead. Alternatively, said straincan be used in the form of a bacterial lysate or in the form ofbacterial fractions; the bacterial fractions suitable for this use canbe chosen, for example, by testing their properties on mercury removalfrom an aqueous environment. Preferably the bacterial cells are presentas living, viable cells.

Food-Grade Bacteria for Removing Toxic Compounds

In one embodiment, the present invention relates to food-grade bacterialor extracts thereof, including probiotics, capable of removing orsequestering toxic compounds from an environment to which the food-gradebacteria is exposed to, or from a substance which may have or may besuspected of having the toxic compound. Substances may include ediblecompositions, such as vegetable-based foods or animal-based foods, andmay also include drinkable solutions, including water, milk, syrups,extracts and other beverages. Substances may also include rawagricultural products used to produce foods and drinkable solutions. Assuch, the present invention relates also to methods of using thefood-grade bacteria of the present invention to prevent the uptake oftoxic compounds by a subject, or in methods to filter toxic compoundsout of substances prior to exposing a subject to said substances. Theenvironment may include an aqueous environment, such as thegastro-intestinal tract of a subject, or the environment in which thesubject resides, such as a pond.

The food grade bacteria may be any type of bacteria that may be capableof removing toxic compounds from foods or solutions that may be consumedby a subject, or from ingredients used in the manufacture of said foodsor solutions. Table 1 includes food-grade bacteria that may be used withthe present invention. In a preferred aspect, the food-grade bacteriamay be aerobically, microaerophilically or anaerobically grown and maybe selected from the group consisting of the food-grade bacteria ofTable 1. Administration of the food-grade bacteria, or extract thereof,to a subject may be accomplished by any method likely to introduce theorganisms into the gastro-intestinal tract of the subject. The bacteriacan be mixed with a carrier and applied to liquid or solid feed or todrinking water. The carrier material should be non-toxic to the subject.When dealing with live food-grade bacteria, the carrier material shouldalso be non-toxic to the food-grade bacteria. When dealing with livefood-grade bacteria the carrier, preferably, may include an ingredientthat promotes viability of the bacteria during storage. The food-gradebacteria may also be formulated as an inoculant paste to be directlyinjected into a subject's mouth. The formulation may include addedingredients to improve palatability, improve shelf-life, impartnutritional benefits, and the like. If a reproducible and measured doseis desired, the food-grade bacteria can be administered by a cannula orsyringe. The amount of food-grade bacteria to be administered isgoverned by factors affecting efficacy. When administered in feed ordrinking water the dosage can be spread over a period of days or evenweeks. The cumulative effect of lower doses administered over severaldays may be greater than a single larger dose thereof. One or morestrains of food-grade bacteria may be administered together. Acombination of strains may be advantageous because individual subjectsmay differ as to the strain which is most persistent in a givenindividual.

The present invention is also directed to extracts or fragments offood-grade bacterial that may be capable of removing or sequesteringtoxic compounds from a substance or sample. As shown herein, theinventors found that dead food-grade bacteria may be used to sequestermercury from a sample. As such the present invention is directed tofood-grade bacteria fragments capable of binding toxic compounds foundin a substance of interest.

Applications

Food-grade bacteria of the present invention may be used as a preventivemeasure, to prevent a subject not presently carrying a toxic compound,from acquiring the toxic compound by exposure to consumables orenvironments where the toxic compounds are present. Food grade bacteriaof the present invention may also be used to substantially reduce orsubstantially eliminate toxic compounds from a subject.

Treatment of a subject carrying the toxic compounds may be accomplishedto reduce or eliminate the amount of the toxic compound carried by thesubject, by administering the food-grade bacteria, or extracts thereof,to the subject carrying the toxic compound.

The methods for administering food-grade bacteria may essentially be thesame, whether for prevention or treatment. By routinely administering aneffective dose to a subject, the risk of contamination by the undesiredtoxin may be substantially reduced or substantially eliminated by acombination of prevention and treatment.

In one embodiment, food-grade bacteria of the present invention may beused in methods to filter toxic compounds out of a substance. Themethod, in one embodiment, may comprise contacting the substance withthe food-grade bacteria for a sufficient amount of time, and removingthe food-grade bacteria and the toxin from the substance. To accomplishthis filtration of toxic compounds from a substance, the food-gradebacteria, extracts or fragments of said food-grade bacteria capable ofbinding to the toxic compounds, may, for example, be attached to afilter, or to a solid support, such as an affinity column, and thesubstance may then be run through the filter or affinity column.

Food-grade bacteria may also be used, according to another embodiment ofthe present invention, to feed aquatic animals such as fish and shrimp.In one embodiment, food-grade bacteria of the present invention may, forexample, be added to tanks and ponds containing the aquatic animal.Preferably the food-grade bacteria used for aquatic animals, may be abacteria that occurs naturally in fresh and sea water environments.

Preparation and Administration

Although this invention is not intended to be limited to any particularmode of application, oral administration of the compositions arepreferred. One food-grade bacterium may be administered alone or inconjunction with a second, different food-grade bacterium. Any number ofdifferent food-grade bacteria may be used in conjunction. By “inconjunction with” is meant together, substantially simultaneously orsequentially. The compositions may be administered in the form oftablet, pill or capsule, for example. One preferred form of applicationinvolves the preparation of a freeze-dried capsule comprising thecomposition of the present invention. Another preferred form ofapplication involves the preparation of a lyophilized capsule of thepresent invention. Still another preferred form of application involvesthe preparation of a heat dried capsule of the present invention.

By “amount effective” as used herein is meant an amount of food-gradebacterium or bacteria, e.g., Lactobacillus, high enough to significantlypositively modify the condition to be treated but low enough to avoidserious side effects (at a reasonable benefit/risk ratio), within thescope of sound medical judgment. An effective amount of Lactobacilluswill vary with the particular goal to be achieved, the age and physicalcondition of the subject being treated, the duration of treatment, thenature of concurrent therapy and the specific Lactobacillus employed.The effective amount of Lactobacillus will thus be the minimum amountwhich will provide the desired detoxification.

A decided practical advantage is that the food-grade bacteria, e.g.Lactobacillus, may be administered in a convenient manner such as by theoral, intravenous (where non-viable), or suppository (vaginal or rectal)routes. Depending on the route of administration, the active ingredientswhich comprise food-grade bacteria may be required to be coated in amaterial to protect said organisms from the action of enzymes, acids andother natural conditions which may inactivate said organisms. In orderto administer food-grade bacteria by other than parenteraladministration, they should be coated by, or administered with, amaterial to prevent inactivation. For example, food-grade bacteria maybe co-administered with enzyme inhibitors or in liposomes. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DFP) and trasylol. Liposomes includewater-in-oil-in-water P40 emulsions as well as conventional andspecifically designed liposomes which transport Lactobacilli or theirby-products to an internal target of a host subject.

The food-grade organisms may also be administered parenterally orintraperitoneally. Dispersions can also be prepared, for example, inglycerol, liquid polyethylene glycols, and mixtures thereof, and inoils.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol, and the like), suitable mixtures thereof and vegetable oils. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion. In many cases it will be preferable to includeisotonic agents, for example, sugars or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating thefood-grade bacteria in the required amount in the appropriate solventwith various of the other ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized food-grade bacteria into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof. Additional preferred methods of preparation include but are notlimited to lyophilization and heat-drying.

When the food-grade bacteria are suitably protected as described above,the active compound may be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or it may beenclosed in hard or soft shell gelatin capsule, or it may be compressedinto tablets designed to pass through the stomach (i.e., entericcoated), or it may be incorporated directly with the food of the diet.For oral therapeutic administration, the food-grade bacteria may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like.

The tablets, troches, pills, capsules, and the like, as described above,may also contain the following: a binder such as gum tragacanth, acacia,corn starch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acid,and the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavoringagent such as peppermint, oil or wintergreen or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills or capsules orLactobacilli in suspension may be coated with shellac, sugar or both.

A syrup or elixir may contain the active compound, sucrose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, thefood-grade organism may be incorporated into sustained-releasepreparations and formulations.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of the food-grade bacteriacalculated to produce the desired preventive or therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the novel dosage unit forms of the invention may be dictated by andmay be directly depending on (a) the unique characteristics of thefood-grade bacteria and the particular preventive, detoxification ortherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such food-grade bacteria for the establishmentand maintenance of a healthy flora in the intestinal tract.

The food-grade organism is compounded for convenient and effectiveadministration in effective amounts with a suitable pharmaceutically orfood acceptable carrier in dosage unit form as hereinbefore disclosed. Aunit dosage form can, for example, contain the principal active compoundin an amount approximating 10⁹ viable or non-viable, e.g., Lactobacilli,per ml. In the case of compositions containing supplementary ingredientssuch as prebiotics, the dosages are determined by reference to the usualdose and manner of administration of the said ingredients.

The pharmaceutically acceptable carrier may be in the form of milk orportions thereof including yogurt. Skim milk, skim milk powder, non-milkor non-lactose containing products may also be employed. The skim milkpowder is conventionally suspended in phosphate buffered saline (PBS),autoclaved or filtered to eradicate proteinaceous and livingcontaminants, then freeze dried heat dried, vacuum dried, orlyophilized.

Some other examples of substances which can serve as pharmaceuticalcarriers are sugars, such as lactose, glucose and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethycellulose, ethylcellulose and cellulose acetates;powdered tragancanth; malt; gelatin; talc; stearic acids; magnesiumstearate; calcium sulfate; calcium carbonate; vegetable oils, such aspeanut oils, cotton seed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,manitol, and polyethylene glycol; agar; alginic acids; pyrogen-freewater; isotonic saline; cranberry extracts and phosphate buffersolution; skim milk powder; as well as other non-toxic compatiblesubstances used in pharmaceutical formulations such as Vitamin C,estrogen and echinacea, for example. Wetting agents and lubricants suchas sodium lauryl sulfate, as well as coloring agents, flavoring agents,lubricants, excipients, tabletting agents, stabilizers, anti-oxidantsand preservatives, can also be present.

Accordingly, the subject may be orally administered a therapeuticallyeffective amount of at least one food-grade bacteria and apharmaceutically acceptable carrier in accordance with the presentinvention. The food-grade bacteria may be a Lactobacillus. TheLactobacillus may be selected from the group comprising the bacterialisted in Table 1.

TABLE 1 Strains Tested For Ability to Degrade or Sequester ToxicCompounds Species Strain Code 1 Strain Code 2 Lactobacillus caseiShirota YIT 9029 FERM BP-1366 Lactobacillus casei ATCC 393 Lactobacilluscrispatus ATCC 33323 Lactobacillus fermentum ATCC 11739 Lactobacillusjohnsonii DSM 20553 Lactobacillus plantarum ATCC 14917 Lactobacillusrhamnosus ATCC 27773 Lactobacillus reuteri RC-14 ATCC 55845Lactobacillus amylovorus LAB Lactobacillus rhamnosus GG ATCC 53013Lactobacillus rhamnosus GR-1 ATCC 55826 Lactobacillus rhamnosus HN001Lactobacillus rhamnosus R37 DN 116-0060 Lactobacillus rhamnosus R38 DN116-0063 Lactobacillus rhamnosus R22 DN 116-0009 Lactobacillus rhamnosusR17 DN 116-0136 Lactobacillus rhamnosus R29 DN 116-0064 Lactobacillusrhamnosus R3 DN 116-0061 Lactobacillus rhamnosus R10 DN 116-0032Lactobacillus rhamnosus R11 DN 116-0141 Lactobacillus casei C3 DN114-0017 Lactobacillus casei C8 DN 114-0022 Lactobacillus casei C11 DN114-0125 Lactobacillus casei C26 DN 114-0074 Lactobacillus casei C6 DN114-0226 Lactobacillus casei C20 DN 114-0037 Lactobacillus casei C29 DN114-0230 Lactobacillus casei C13 DN 114-0126 Lactobacillus casei C28 DN114-0189 Lactobacillus casei C31 DN 114-0227 Lactobacillus casei C10 DN114-0223 Lactobacillus casei C1 DN 114-0001

The above disclosure generally describes the present invention. Changesin form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

EXAMPLES

The examples are described for the purposes of illustration and are notintended to limit the scope of the invention.

Example 1—Demonstration of Removal of Inorganic Lead and Cadmium from anAqueous Environment

1 mL inoculums of 24 hour cultures of Lactobacillus rhamnosus GR-1,Lactobacillus casei 393T, Lactobacillus johnosonii 20553 andLactobacillus plantarum 14917T at cell concentrations of approx. 1×10⁹CFU/mL were added to a 50 mM HEPES buffer containing Pb or Cd andincubated for 5 hours at 37° C. Following incubation, cells were removedby centrifugation at 5, 000 G. The total metal concentration in thesupernatant was analyzed via Inductively Coupled Plasma-MassSpectrometry (ICP-MS).

FIGS. 1A-1B illustrate the ability of food grade Lactobacilli to removePb (FIG. 1A) and Cd (FIG. 1B) from a solution at starting inoculums of 2ppm and 2.5 ppm for lead and cadmium respectively. Depending on thespecies/strain of Lactobacilli examined and the metal environment therewas variation in removal. As illustrated in FIG. 1A 45-50% of Pb wasremoved from solution while as illustrated in FIG. 1B 40-80% of Cd wasremoved. Removals of both Pb and Cd were deemed significant (p<0.05) byan ANOVA one-way analysis of variance.

Example 2—Demonstration of Specificity of Lead and Cadmium Removal byFood Grade Lactobacilli from an Aqueous Solution

1 mL inoculums of 24 hour cultures of Lactobacillus rhamnosus GR-1,Lactobacillus rhamnosus GG, E. coli Col and E. coli 25922 at cellconcentrations of approx. 1×10⁹ CFU/mL were added to a 50 mM HEPESbuffer containing Pb or Cd and incubated for 5 hours at 37° C. Followingincubation, cells were removed by centrifugation at 5, 000 G. The totalmetal concentration in the supernatant was analyzed via InductivelyCoupled Plasma-Mass Spectrometry (ICP-MS). As illustrated in FIGS.2A-2B, for both Pb (FIG. 2A) and Cd (FIG. 2B), Lactobacilli removed70-80% of metal in solution while E. coli removal was only 30-50%. Theamount removed by Lactobacilli compared to E. coli strains anduninoculated control were shown to be significant (P<0.05) by an ANOVAone-way analysis of variance.

Example 3—Removal of Lead and Cadmium by Live and Dead Lactobacilli

In this example, the ability of live and dead Lactobacilli to removelead (FIG. 3A) cadmium and (FIG. 3B) from solution at a startinginoculums of 3 ppm was tested.

The assay was carried out as previously described in Examples 1 and 2.Viable cells of all Lactobacilli were compared to cells that were killedby gamma irradiation at 5.5 Kilo Grays (KG) for 1 hr. Gamma irradiationwas used as it kills the cells without destroying cell wall/membraneintegrity. Equal inoculums of viable and dead cells were used. Withreference to FIG. 3B, live and cells irradiated with gamma rays wereable to remove roughly equal amounts of cadmium. However, as illustratedin FIG. 3A, there was a split between the ability of viable or deadcells to bind more lead. The results obtained herein show that bindingof metals may likely be a surface associated action not requiringactively metabolic cells. As such, the present invention is alsodirected to the parts of food-grade bacteria capable of binding heavymetals.

Example 4—Demonstration of Passive Sequestration Activity

FIGS. 4A-4C illustrate TEM micrographs of Lactobacillus rhamnosus R37incubated in 50 mM HEPES-NaOH buffer (FIG. 4A) with 1 mM Pb (FIG. 4B)and 1 mM HgCl₂ (FIG. 4C) added. Numerous deposits are observedthroughout the cells incubated with heavy metals (FIGS. 4B-4C) however;some smaller deposits are also visible when no metal is added (FIG. 4A).The nature of the deposits was confirmed using SEM and EDX analysis.

FIGS. 5A-5C are SEM micrographs of Lactobacillus rhamnousus R37incubated in 50 mM HEPES-NaOH buffer (FIG. 5A) with 1 mM Pb (FIG. 5B)and 1 mM HgCl₂ (FIG. 5C) added. Numerous deposits are observedthroughout the cells incubated with heavy metals (FIGS. 5B-5C) however;some smaller deposits are also visible when no metal is added (FIG. 5A).

FIGS. 6A-6C illustrate energy-dispersive X-ray spectroscopy (EDX)analysis of putative metal deposits in Lactobacillus rhamnosus R37.Osmium coated samples being imaged with SEM were analyzed with EDX todetermine the elemental composition of putative metal deposits withinthe cell. FIG. 6A demonstrates the spectrum (bottom) of a portion ofcell not containing any visible deposits and mercury was not detected.FIG. 6B shows analysis of a large deposit which was determined tocontain 36.62% mercury by mass proving cellular sequestration of mercury(see Table 2).

Similar results were also obtained for GR-1, R3, R39, Lactobacilluscasei C3 showing mercury in the cell.

TABLE 2 Control Suspected Hg deposit Element Weight % Atomic % ElementWeight % Atomic % Carbon 74.44 87.03 Carbon 39.67 70.99 Oxygen 11.7510.32 Nitrogen 7.51 11.52 Sulfur 4.50 1.97 Oxygen 8.51 11.44 Osmium 9.300.69 Phosphorus 1.23 0.85 Totals 100.00 100.00 Sulfur 0.97 0.65 Osmium5.50 0.62 Mercury 36.62 3.92 Totals 100.00 100.00

Example 5—Confirmation of Precipitation and Binding of Metals on andwithin Food Grade Bacteria

Lactobacilli were incubated in a 50 mM HEPES buffer for 2 hrs at 37° C.in the presence of metals at a final concentration of 10 mM. The assaywas carried out by incubating bacteria (Lactobacillus rhamnousus GR-1)for 2 hrs in a 10 mM metal solution at 37° C. Following incubation thebacteria were diluted 100-fold and filtered through a 0.2 μm filter totrap bacteria and allow passage of solution. The filters were dried atroom temperature for 2 hrs and then coated with 5 nm of osmium tetraoxide. The identification of the metals was confirmed by EDAX X-rayanalysis which showed that the metal precipitates were the heavy metalsadded to solution.

FIGS. 7A-7C are scanning electron micrographs (SEM) of Lactobacillusrhamnosus GR-1 incubated with (FIG. 7A) lead or (FIG. 7B) cadmium. Thebright spots observable in the images represent the precipitation ofheavy metal particles on the surface and inside the cell. FIG. 7Cdisplays the non metal control which is the Lactobacilli withoutaddition of metals, note the absence of precipitate metal particles.

Example 6—Preliminary Evidence of Protective Effect of Food GradeLactobacilli on a Caco-2 Cell Line as a Model of the Gut EpithelialBarrier

Caco-2 cells were grown in 12 or 24 well plates for two weeks usingsupplemented Eagles Minimum Essential Medium (ATCC®) as described above.At two weeks, media was aspirated and cells were washed lightly 2× withwarm 50 mM HEPES buffer. Bacterial cultures of interest were also grownin 5 mL broth cultures for 22 hrs and washed 2× with 50 mM HEPES.Bacterial cells were resuspended to 10 mL in Eagles Minimum EssentialMedium (ATCC®) without any Pen/Strep in solution, 400 μL of media wasadded to wells in 24 well plates and 900 μL of media was used in 12 wellplates. Bacteria were allowed to incubate with cell line for 2 hr at 37°C. During incubation period metal spiked solutions of Eagles MinimumEssential Medium (ATCC®) was made by adding stock concentrations of Pb,Cd or As (Sigma Aldrich®) to the media at desired concentrations.Following incubation period the bacterial metal solution was aspiratedso that only cells adhering to the Caco-2 cell monolayer remained, themedia was replaced with the metal spiked media in addition control wellswere set up that either did not have metal in the media and were notincubated with bacterial species. Cells were incubated for 5 hrs inmetal spiked media at 37° C.

Following this incubation, media was removed by aspiration anddiscarded. Cells were washed once gently with warm HEPES buffer and thenremoved from the wells using 500 ul of 0.25% (w/v) trypsin until cellsdetached from flask. 500 μL of cell media was added to stop trypsinreaction and total volume of each well was transferred into separatesterile 1.5 mL centrifuge tubes (Diamed®). The cell suspension was mixedby pipetting to avoid formation of bubbles. Cells were centrifuged in abench top microcentrifuge for 2 mins at 120 RPM, supernatant wasdiscarded and cells were suspended in 1×PBS. Cells were diluted by afactor of 10 by suspending 50 μL of cells with 450 μL of Guava Viacount®Reagent (Cat No. 4000-0041) in a clean sample tube, cells were stainedfor at least 5 min. Stained cells were then analyzed for viability usingthe Guava ViaCount Assay on the Guava EasyCyte Mini bench topflowcytometer. Cells were separated based on viability forming twodistinct populations: live and dead. Populations were analyzed andstatistically compared using FlowJo (TreeStar™) analysis software forflow cytometry data. Cells were analyzed to see differences in viabilityafter exposure to metals in the presence or absence of Lactobacilli.

FIG. 8 illustrates a flow cytometry analysis of the Caco-2 cell linecomparing viability vs. mortality for un treated cells (FIG. 8A), Caco-2cells exposed to cadmium (FIG. 8B), Caco-2 cells exposed toLactobacillus plantarum 14917T (FIG. 8C) and Caco-2 cells pretreatedwith Lactobacillus plantarum 14917T and then exposed to cadmium (FIG.8D). As shown by FIG. 8D addition of Lactobacillus plantarum 14917Tbefore cadmium exposure contributed to increased survival of the cellline then when just exposed to cadmium (FIG. 8B).

Example 7—Viability of Lead and Cadmium Resistant Food Grade Bacteria ofthe Genus Lactobacillus

The assay was carried out by inoculating a 200 μL well of Man RogosaSharpe (MRS) medium containing lead or cadmium at a concentration of 100ppm with an inoculum of 10⁷ bacteria from a fresh 24 hrs broth culturesof the Lactobacilli species Lactobacillus rhamnosus GR-1 andLactobacillus plantarum 14917T. Growth was measured by OD600 for 24 hrs.incubation at 37° C. Growth was measured for 24 hours with readingstaken every 30 minutes by optical density measurements at a wavelengthof 600 nm. Following the growth assay all species were diluted and dropplated on MRS agar to determine colony forming units (CFU) in solution.

FIGS. 9A-9B show growth of all tested Lactobacilli species in the MRSmedia with lead (FIG. 9A) or cadmium (FIG. 9B) at a concentration of 100ppm.

Example 8—Demonstration of Removal of Inorganic Mercury from an AqueousEnvironment

A 1% inoculum of a 24 hour culture of Lactobacillus rhamnosus DN116-060was added to de Man Rogosa Sharpe (MRS) broth containing HgCl₂ andincubated for 24 hours at 37° C. Following incubation, cells wereremoved by centrifugation at 5,000 g. The total mercury concentration inthe supernatant was analyzed via cold vapor atomic absorptionspectroscopy (CVAAS). As illustrated in FIGS. 10A-10B, the Lactobacilliremoved 94.4% of a 1 part per million (ppm) mercury inoculum (FIG. 10A)and 85% of a 15 part per billion (ppb) inoculum (FIG. 10B). Bothremovals were deemed significant (p<0.05) by an unpaired T-test.

Example 9—Demonstration of Removal of Organic Mercury Form an AqueousEnvironment

A 1% inoculum of a 24 hour culture of Lactobacillus rhamnosus DN116-060was added to de Man Rogosa Sharpe (MRS) broth containing MeHgCl2 andincubated for 24 hours at 37° C. Following incubation, cells wereremoved by centrifugation at 5,000 g. The total mercury concentration inthe supernatant was analyzed via cold vapor atomic absorptionspectroscopy (CVAAS).

FIG. 11 shows the ability of a food grade bacterium to remove MeHg²⁺from solution at a starting inoculum of 1 ppm MeHgCl2. (Error bars±SEM).As illustrated in FIG. 11, the Lactobacilli removed 23.2% of a 1 ppmmercury inoculum (p<0.05 by an unpaired t-test).

Example 10—Inorganic Mercury Removal by Live and Dead Lactobacillusrhamnosus DN116-060

The assay was carried out as previously described in Example 9 at aconcentration of 500 ppb HgCl₂. Viable cells of Lactobacillus rhamnosusDN116-010 were compared to cells that were killed by heating at 80° C.for 10 minutes at an inoculum equivalent to the final cell density ofviable cells.

FIG. 12 illustrates the ability of live and dead Lactobacillus rhamnosusDN116-060 to remove Hg²⁺ from solution at a starting inoculum of 500 ppbHgCl₂. As shown in FIG. 12, viable cells were capable of removingsignificantly more mercury than heat killed cells (p<0.05 by unpairedt-test) suggesting that there is a passive sequestering of mercury aswell as potential metabolic detoxification.

Example 11—Variability of Mercury Resistance within Food Grade Bacteriaof the Genus Lactobacillus

Assay was carried out as previously described in Example 9 across aspectrum of HgCl₂ concentrations. Growth was measured after 24 hours at37° C. by the optical density of cultures at a wavelength of 600 nm. Aspectrum of resistances to mercury were observed in both speciesdemonstrating that resistance to mercury is a variable trait among foodgrade bacteria.

FIGS. 13A-13B illustrate the growth of Lactobacillus casei (n=38) (FIG.13A) and Lactobacillus rhamnosus (n=40) (FIG. 13B) in a gradient of Hg²⁺measured by OD600 after 24 hours incubation at 37° C. Each set ofconnected points represents one strain. Resistance is a strain variabletrait resulting in a spectrum of resistance profiles in both species.FIG. 13B illustrates three Lactobacillus rhamnosus strains showing adistinctly higher resistance as compared to the rest of the strains.

Example 12

Twenty-four hour time course of mercury removal by Lactobacillusrhamnosus R37 (in viable and heat killed form) and GR-1 in HEPES-NaOHsupplemented with 1 μg/mL HgCl₂ incubated at 37° C. With reference toFIG. 14, sequestration activity is not instantaneous and reaches amaximum after 12 h in Lactobacillus rhamnosus R37 while maximal removalwas observed at 24 hours in the case of Lactobacillus rhamnosus GR-1.

Example 13—Resistant Strains of Food Grade Bacteria Remove More Mercurythan Mercury Sensitive Strains

The assay described in Example 1 was carried out using a selection ofLactobacillus rhamnosus strains of increased resistance and increasedsensitivity to mercury.

FIGS. 15A-15B illustrate removal of mercury from solution by a selectionof Lactobacillus rhamnosus strains of increased resistance (R) andstrains of increased sensitivity (S) to mercury at HgCl₂ concentrationsof 0.5 ppm (FIG. 15A) and 1 ppb (FIG. 15B). Resistant strains removedsignificantly more mercury from solution than their sensitivecounterparts (p<0.05 as determined by ANOVA with Bonferroni post test[FIG. 15A] and un-paired t-test [FIG. 15B]). (Error bars±SEM)

Example 14—Removal of Arsenite and Arsenate from an Aqueous Environment

Bacterial cultures were grown for 24 hrs in preferential media; ManRogoas Sharpe (MRS) broth for Lactobacilli and Luria-Bertani (LB) brothfor E. coli. Cells were centrifuged, washed and re-suspended in PBS. 1mL aliquouts were distributed between sample tubes containing 9 mL ofPBS buffer spiked with arsenic, 1 mL of MRS or LB broth was added tosample tubes. Cells were incubated for 5 hrs at 37° C.; followingincubation cells were removed by centrifugation at 5, 000 g. The totalarsenic remaining in solution was analyzed via inductively coupledplasma-mass spectrometry (ICP-MS). As illustrated in FIG. 16Lactobacilli were able to remove 50-60% of As (III) and As (V) while E.coli DH5a was less effective.

Example 15—Demonstration of Removal of Arsenite (as III) by a Panel ofLactobacilli

The assay was carried out by inoculating a 1 ppm (9.08×1018 free atoms)arsenite solution (HEPES buffer) with 1×10⁹ CFU/mL of selectedLactobacilli. The solutions were incubated for 5 hrs at 37° C.;following incubation cells were removed by centrifugation at 5, 000 g.The total arsenic remaining in solution was analyzed via inductivelycoupled plasma-mass spectrometry (ICP-MS).

As shown in FIG. 17 and Table 3, Lactobacilli removed 11-13% of thetotal arsenic which was determined by looking at differences inconcentrations in total free atoms in solution vs. bound to eachspecies.

TABLE 3 Species % Removed L. rhamnosus GR-1 13 L. johnsonii 20553 11 L.casei 393T 11 L. plantarum 14917T 11

Example 16—Demonstration of Removal of Arsenic (III) at HighConcentrations by Lactobacilli

The assay was carried out by inoculating a 100 ppm arsenite solution ofHEPES buffer with 1×10⁹ CFU/mL of the selected Lactobacilli. Thesolutions were incubated for 5 hrs at 37° C.; following incubation cellswere removed by centrifugation at 5,000 g. The total arsenic remainingin solution was analyzed via inductively coupled plasma-massspectrometery (ICP-MS).

As shown in FIG. 18, all Lactobacilli showed ability to remove near 70%of arsenic from solution compared to the untreated control sample.Species to species variation in amount of arsenic able to remove was lowand not significant.

Example 17—Demonstration of Removal of Malathion and Parathion fromAqueous Environment by Probiotic Bacteria

Bacterial broth cultures of Lactobacillus rhamnosus GR-1 were grown for24 hrs in Man Rogosa Sharpe (MRS) broth. Cells were collected, washedand re-suspended in a 1×PBS buffer. 1 mL of cell suspension wastransferred into sample tube containing a 50:50 mixture of HEPES bufferhaving the pesticides and MRS. Starting inoculums of pesticides formalathion and parathion was 5 μg/L of HEPES buffer and 0.5 μg/L of HEPESbuffer respectively. Samples were incubated for 5 hrs at 37° C.Following incubation cells were removed by centrifugation at 5, 000 g.The remaining pesticide in solution was analyzed via gaschromatography-mass spec (GC-MS) and values were compared to untreatedcontrols.

With reference to FIGS. 19A-19B, Lactobacillus rhamnosus GR-1 was ableto remove 20% of the malathion from solution (FIG. 19A) and 50% of theparathion (FIG. 19B).

Example 18—Demonstration of Removal of Malathion and ParathionSimultaneously by a Probiotic Bacterium

Bacterial broth cultures of Lactobacillus rhamnosus GR-1 were grown for24 hrs in Man Rogosa Sharpe (MRS) broth. Cells were collected, washedand re-suspended in a 1×PBS buffer. 1 mL of cell suspension wastransferred into sample tube containing a 50:50 mixture of HEPES bufferhaving the pesticides and MRS. Starting inoculums of pesticides formalathion and parathion was 5 μg/L of HEPES buffer and 0.5 μg/L of HEPESbuffer respectively. Samples were incubated for 5 hrs at 37° C.Following incubation cells were removed by centrifugation at 5, 000 g.The remaining pesticide in solution was analyzed via gaschromatography-mass spec (GC-MS) and values were compared to untreatedcontrols.

As shown in FIG. 20, Lactobacillus rhamnosus GR-1 was able to remove 50%of the malathion from solution and 50% of the parathion.

Example 19—Demonstration of Removal of Pesticides by a Panel of FoodGrade Bacteria and Some E. coli Species

Bacterial broth cultures of Lactobacilli were grown for 24 hrs in ManRogosa Sharpe (MRS) broth, E. coli species were grown for 24 hours inLucella Broth (LB). Cells were collected, washed and re-suspended in a1×PBS buffer. 1 mL of cell suspension was transferred into sample tubescontaining a 50:50 mixture of HEPES buffer having the pesticide and MRSor LB. Starting inoculums of pesticides for malathion and parathion was10 mg/L of HEPES buffer and 3 mg/L of HEPES buffer respectively. Sampleswere incubated for 5 hrs at 37° C. Following incubation cells wereremoved by centrifugation at 5, 000 g. The remaining pesticide insolution was analyzed via gas chromatography-mass spec (GC-MS) andvalues were compared to untreated controls.

FIG. 21A illustrates that the Lactobacilli were able to remove 35-60% ofmalathion, while E. coli was able to remove 10-25% of malathion. FIG.21B illustrates that the Lactobacilli and E. coli were able to remove55-70% of parathion.

Example 20—Demonstration of Removal of Endotoxins by a Panel of FoodGrade Bacteria

Endotoxins are well known toxins responsible for sepsis and death. Theyare produced by a number of Gram negative bacteria and to date feweffective treatments have been developed. Other potent toxins producedby bacteria include the fatal Shiga toxin produced by E. coli 0157:H5,and TcdA and TcdB toxins from Clostridium difficile both of which damagethe human colonic mucosa and are potent cytotoxic enzymes. Deaths fromC. difficile toxins have become a major concern in North Americanhospitals and care homes. Probiotic therapy has shown great promise inpreventing infections caused by E. coli 0157:H7 and C. difficile.

Alkaline phosphatase levels (activity and protein) can be measured infeces and blood as it has been shown that up-regulation of this enzymecan detoxify endotoxins in the gut and improve gut permeability. A pigmodel is used for this assay. C. difficile toxins will be detected fromstool by a commercially available enzyme-linked fluorescenceimmunoassay.

Example 21—Demonstration of Removal of Aflatoxin by a Panel of FoodGrade Bacteria

Aflatoxin (a hepatic carcinogen) is important contributors to disease,albeit risk of exposure to the mainstay population in N. America is low.Aflatoxin B1 has been included because probiotics can have an effectagainst it, and such results have implications for many sub-populationsin the US (eg large farming communities) and beyond (eg Middle East,Argentina).

The aflatoxin will be measured from blood by affinity column cleanup andLC-MS/MS fluorescence.

Example 22—Demonstration of Removal of Heterocyclic Aromatic Amines(HAA) by a Panel of Food Grade Bacteria

Heterocyclic aromatic amines (HAA) are found in food (eg processed meat)and cause diet-related mutagenesis which plays an etiologic role inchronic diseases, including cardiovascular disease and cancer. Theirdirect association with cancer is low, but the potential for them to beinhibited by probiotics makes them worth studying, as a positive detoxeffect provides a good consumer message.

They will be measured from urine and blood samples using HPLC.

Example 23—Demonstration of Removal of Acrylamide by a Panel of FoodGrade Bacteria

Acrylamide is made industrially but is highly regulated due to itsneurotoxicity. It naturally forms in certain foods, particularlyplant-based foods that are rich in carbohydrates and low in protein,during processing or cooking at high temperatures (French fries, potatochips). Also found heavily in cigarette smoke. Acrylamide is monitoredand studied by Health Canada, but no levels have been established onwhat is toxic/safe, so it's tough to set a ‘limit’ or even tell in astudy what would be considered dangerous. It has a link to causingcancer and information on how much will cause this effect is not known.

Acrylamide will be detected by HPLC.

1.-35. (canceled)
 36. A method for reducing a subject's uptake of toxic compounds consumed by the subject, the method comprising administering to the subject a Lactobacillus capable of sequestering the toxic compound consumed by the subject wherein the Lactobacillus is selected from: Lactobacillus reuteri RC-14, Lactobacillus casei 21052, Lactobacillus casei 393T, Lactobacillus rhamnosus GR-1, Lactobacillus rhamnosus R3, Lactobacillus rhamnosus R37, Lactobacillus johnsonii 20553, Lactobacillus plantarum 14917T, or any combination thereof.
 37. The method of claim 36, wherein the toxic compound is selected from the group consisting of: lead, cadmium, arsenic, malathion and parathion.
 38. The method of claim 36, wherein the Lactobacillus is provided in a viable form.
 39. The method of claim 36, wherein the Lactobacillus is provided in a non-viable form.
 40. The method of claim 36, wherein the Lactobacillus is provided as an extract.
 41. The method of claim 36, wherein the Lactobacillus comprise a combination of two or more different strains of Lactobacillus.
 42. The method of claim 36, wherein the lactobacillus is provided in a composition comprising the Lactobacillus and a suitable carrier.
 43. The method of claim 42, wherein the carrier is a milk-based product.
 44. The method of claim 42, wherein the composition comprises a combination of two or more strains of Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus amylovorus.
 45. A method for reducing in a subject gastrointestinal uptake of toxic compounds consumed by the subject through edible or drinkable substances contaminated with the toxic compounds, the method comprising administering to the subject a Lactobacillus, wherein the Lactobacillus is selected from: Lactobacillus reuteri RC-14, Lactobacillus casei 21052, Lactobacillus casei 393T, Lactobacillus rhamnosus GR-1, Lactobacillus rhamnosus R3, Lactobacillus rhamnosus R37, Lactobacillus johnsonii 20553, Lactobacillus plantarum 14917T, or any combination thereof.
 46. The method of claim 45, wherein the toxic compound is selected from the group consisting of: lead, cadmium, arsenic, malathion and parathion.
 47. The method of claim 45, wherein the Lactobacillus is provided in a viable form.
 48. The method of claim 45, wherein the Lactobacillus is provided in a non-viable form.
 49. The method of claim 45, wherein the Lactobacillus is provided as an extract.
 50. The method of claim 45, wherein the Lactobacillus comprise a combination of two or more different strains of Lactobacillus.
 51. The method of claim 45, wherein the lactobacillus is provided in a composition comprising the Lactobacillus and a suitable carrier.
 52. The method of claim 51, wherein the carrier is a milk-based product.
 53. The method of claim 51, wherein the composition comprises a combination of two or more strains of Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus amylovorus. 